Most agriculturally important crop plants are propagated by seed. The seed is planted and under favorable environmental conditions, the seed germinates and grows into crop plants. However, frequently conditions can occur in which after planting of the seed, the seed fails to germinate or germinates poorly producing a thin stand of plants with reduced yield or necessitating the replanting of the crop with new seed at considerable expense to the grower. This has been shown to occur with soybean, corn, and canola in wet and cool field conditions (Wang et al., Enviro. Exp. Bot. 36: 377–383, 1996; Zheng et al., Crop Sci. 34: 1589–1593, 1994). It is often necessary to plant more seeds than predicted to be necessary to achieve a good crop. The percent of seeds that germinate is considered good at 80%. A measurable savings in resources can be achieved if the seed germination can be controlled to achieve 90% or greater seed germination and vigorous seedling growth. Also, seeds may germinate precociously if the environmental conditions at crop maturity are such that the seed prematurely sprout. This is a problem in some wheat varieties and causes a loss of yield and quality of the harvested grain. Dormancy of seeds during storage is an important criteria for a quality product. Adequate storage and shipping characteristics of seeds is an important prerequisite for distributing food products around the world. In many developing countries storage facilities are inadequate and seed and food quality may be affected when seed dormancy is broken and the process of seed germination begins in storage. However, seeds that are chemically treated to inhibit seed germination often show characteristic traits such as reduced plant height and seedling vigor for some period of time after germination. Seed, genetically engineered for a high level of seed dormancy, can be stored more efficiently and suffer fewer side effects than chemically treated germination inhibition.
The failure of seeds to germinate uniformly and at high frequency is an important factor affecting crop yield. Soybean (Glycine max) is a crop species that suffers from loss of seed germination during storage and fails to germinate when soil temperatures are cool. It has been shown that the exogenous treatment of gibberellic acid will stimulate soybean seed germination under conditions that the seed will not normally germinate (Zhang et al., Plant Soil 188: 329–335, 1997). Sugar beet (Beta vulgaris) seed is often chemically treated to improve germination and plant stand which has a direct affect on the yield of the crop. Canola seed germination and seedling growth can be improved at low temperatures by treatment with gibberellic acid (Zheng et al., Crop Sci. 34: 1589–1593, 1994). Improved seedling vigor is observed by these treatments with the plants emerging more quickly from the soil and are more likely to establish themselves under adverse environmental conditions.
There is a need for an effective system which would couple genetically improved seed dormancy with a chemical seed treatment to induce seed germination when germination is desired. The genetic control of gibberellin activity in developing seeds, germinating seeds and during early seedling growth coupled with exogenous replacement of the activity would be an effective means to control seed germination and seedling growth.
Inhibitors of gibberellin biosynthesis suggest that de novo synthesis of GA is a prerequisite for the release from dormancy (Thomas, Plant Growth Reg. 11: 239–248, 1992). A key enzyme of gibberellin biosynthesis is copalyl diphosphate synthase (CPS) (formerly ent-kaurene synthetase (EKS-A)). Two enzymes, CPS and KS-B (ent-kaurene synthetase-B), catalyze the cyclization of geranylgeranyl diphosphate to ent-kaurene. CPS is the first committed step in GA biosynthesis. Plant mutants blocked at CPS show strong adverse germination/seedling vigor phenotypes that can be reversed by the application of an exogenous supply of GA. Although these mutants demonstrate the role of GA in seed germination, they do not establish the developmental timing required for expression of GA for normal seed germination and seedling growth. It has not been previously established that soybean plants require de novo biosynthesis of GA for normal seed germination and early seedling growth. It has also not been previously demonstrated that endogenous levels of GA can be affected by the expression of an antisense RNA to a gene important in the biosynthesis of GA in soybean. There are no known soybean mutants blocked in GA biosynthesis; therefore, the requirement for de novo GA biosynthesis in soybean is unknown. Inhibitors of GA biosynthesis offer a method to investigate the effect of decreased GA biosynthesis on soybean germination and seedling growth. GA biosynthesis inhibitors can block ent-kaurene biosynthesis or can block at ent-kaurene oxidation or can inhibit the late dioxygenase-catalyzed steps (Jung et al., J. Plant Growth Regul. 4: 181–188, 1986).
Dioxygenase enzymes modify various gibberellin substrates. The dioxygenases, 20-oxidase and 3β-hydroxylase, are involved in the biosynthesis of GA precursors and active forms. The overexpression or suppression of the GA 20-oxidase genes affect seedling growth differentially (Hedden, et al., In Genetic and environmental manipulation of horticultural crops. Cockshull, Gray, Seymour and Thomas eds. CAB International 1998). Degradation of bioactive GA in specific tissues of the developing seed, germinating seed and early seeding growth can also regulate GA tissue responses. Genes from Arabidopsis and Phaseolus coccineus have been identified that encode for enzymes that have gibberellin 2-oxidase activity (Thomas et al., Proc. Natl. Acad. Sci. U.S.A. 96: 4698–4703, 1999).
Pathways which use substrates in common with the gibberellin pathway are known. The carotenoid pathway (Encyclopedia of Plant Physiology. Secondary Plant Products Vol 8: 259, Bell and Charlwood eds.), the phytol pathway (Encyclopedia of Plant Physiology. Secondary Plant Products Vol 8: 207, Bell and Charlwood eds. ) and the gibberellin pathway each use geranylgeranyl pyrophosphate as a key precursor to the synthesis of their respective end products.
The methods of plant biotechnology provide means to express gene products in plants at particular developmental plant growth stages. Gene promoters that express during seed germination and early seedling development are a preferred embodiment of this invention. The present invention provides a method to genetically suppress seed germination and early seedling development, then by necessity restore normal germination with exogenous application of GA compounds to the seed or seedling. The present invention provides genetically-engineered gibberellin-deficient plants. In agriculture, there exists a need for improved materials and methods for the control of seed germination and seedling growth.